Article pubs.acs.org/jnp
Melodinines M−U, Cytotoxic Alkaloids from Melodinus suaveolens Ya-Ping Liu,†,‡ Yan Li,† Xiang-Hai Cai,† Xing-Yao Li,†,‡ Ling-Mei Kong,†,‡ Gui-Guang Cheng,†,‡ and Xiao-Dong Luo*,† †
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China S Supporting Information *
ABSTRACT: Nine new alkaloids, melodinines M−U (1−9), and 11 known alkaloids were isolated from Melodinus suaveolens. The new structures were elucidated by extensive NMR and mass spectroscopic analyses and comparison to known compounds. All compounds were evaluated for their cytotoxicity against five human cancer cell lines. Compounds 6, 11, and 16 showed significant cytotoxicity.
P
lants of the family Apocynaceae have been proven to be good resources of monoterpenoid indole alkaloids. These originate from condensation of tryptophan with secologanin to give strictosidine, which then elaborates to give an impressive array of structural variants.1 Many of them, such as yohimbine,2 reserpine,3 and camptothecin,4 are well known for their pharmacological significance. Previous pharmacological investigations on the crude and purified alkaloids from some Melodinus plants have demonstrated promising antitumor5 and antibacterial activities.6 Our previous studies reported the isolation and cytotoxic activities of melohenines A and B, melotenine A, and melodinines A−L from two species of Melodinus.7 Continuation of our study on the genus Melodinus has led to the isolation of seven new monoterpenoid indole alkaloids, melodinines M−S (1−7), two new monoterpenoid quinoline alkaloids, melodinines T and U (8, 9), and 11 known alkaloids from Melodinus suaveolens Champ. ex Benth (Apocynaceae).8 To the best of our knowledge, compound 1 is the first Aspidosperma-type alkaloid possessing a dienone ring A. Compounds 2 and 3 are Aspidosperma-type alkaloids each bearing a methyl group at C-10, a type seldom reported previously. Structures of the new alkaloids were elucidated by spectroscopic methods, while the known alkaloids were identified as 11-hydroxytabersonine (10),9 11-methoxytabersonine (11),10 tabersonine (12),11 19(R)-acetoxytabersonine (13),12 11-methoxy-19-(R)-hydroxytabersonine (14),13 lochnericine (15),14 3α-acetonyltabersonine (16),15 3-oxo-tabersonine,16 venalstonine,17 scandine,18 and 10-hydroxyscandine19 by comparison with data (1D NMR and MS) in the literature. All of the compounds were evaluated for cytotoxicity against five human cancer cell lines.
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11 degrees of unsaturation. The IR absorption bands at 3431, 1656, and 1610 cm−1 suggested the presence of a β-anilinoacrylate
RESULTS AND DISCUSSION Melodinine M (1) was isolated as yellow needles and gave a positive reaction with Dragendorff’s reagent. Its molecular formula was established as C21H24N2O4 by the molecular ion at m/z 369.1811 [M + H]+ in the HRESIMS, indicating © 2012 American Chemical Society and American Society of Pharmacognosy
Received: November 19, 2011 Published: January 19, 2012 220
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system,20 corresponding to carbon signals characteristic of an acrylate double bond at δC 158.5 (C-2) and 98.6 (C-16). The 1 H NMR spectrum displayed two olefinic signals at δH 5.80 (ddd, J = 10.2, 4.8, 1.2 Hz) and 5.62 (d, J = 10.2 Hz), which were ascribed to protons of a double bond between C-14 and C-15. An indolic −NH proton was also observed at δH 9.32 (1H, s) (Table 1). The 13C NMR and DEPT spectra of
OH-8, H-21, and H-19. Thus, the structure of melodinine M (1) was established as shown. Melodinine N (2) had the molecular formula C22H26N2O3 as established by HRESIMS. The UV spectrum showed absorption maxima characteristic of a β-anilinoacrylate chromophore (333, 255, and 203 nm), while the IR spectrum showed absorption bands due to −NH (3439 cm−1) and conjugated ester (1669 cm−1) functions.20 The 1D (Tables 1 and 3) and 2D NMR data of 2 were similar to those of 10 except for one more methyl (Me-22) substituted at C-10, as supported by the HMBC correlations of δH 2.08 (3H, s) with δC 123.8 (d, C-9), 120.4 (s, C-10), and 155.0 (s, C-11). ROESY correlations of H9/H-21 and H-21/H-19 indicated that the relative configuration of 2 was also the same as that of 10. Analysis of 2D NMR data (HSQC, HMBC, ROESY) established the structure of 2 to be as shown, and it was named melodinine N. Melodinine O (3) showed almost the same NMR spectra as 2, except for a methoxy in 3 instead of a hydrogen (Me-22) in 2, as indicated by HMBC correlations from δH 3.33 (3H, s) to δC 71.0 (t, C-22) and from δH 4.43 (2H, s, H-22) to δC 123.0 (d, C-9), 116.9 (s, C-10), and 156.5 (s, C-11). Other parts of the structure were identical to those of 2 by detailed analysis of 2D NMR data. Melodinine P (4) was isolated as a colorless oil. The UV spectrum showed absorption maxima characteristic of a β-anilinoacrylate chromophore (342, 313, and 204 nm), and the IR spectrum showed absorption bands due to −NH (3427 cm−1) and conjugated ester (1669 cm−1) functions. The HREIMS gave the molecular formula C21H24N2O3, identical to that of 11hydroxytabersonine (10). The NMR data also suggested that 4 might be the same as 10. Detailed comparison of their 1D NMR data suggested that the OH group was at C-10 in 4, rather than at C-11 in 10, which was further supported by the HMBC correlations of H-9 (δH 6.74, d, J = 2.4 Hz), H-11 (δH 6.62, dd, J = 7.8, 2.4 Hz), and H-12 (δH 6.86, d, J = 7.8 Hz) with C-10 (δC 152.8, s). Analysis of 2D NMR data confirmed that the other parts were the same as those of 10. Hence, the structure of melodinine P (4) was determined as shown. The molecular formula of melodinine Q (5) was determined to be C24H26N2O3 by the positive HRESIMS. The IR spectrum suggested the presence of −NH (3440 cm−1), carboxyl (1718 cm−1), and double-bond (1640, 1614 cm−1) groups. The 13 C NMR and DEPT spectra of 5 displayed 24 carbon resonances ascribed to two methyl, five methylene, nine methine, and eight quaternary carbons (Table 3). These data resembled those of venalstonine.17 One visible difference was that 5 possessed three additional carbon signals at δC 31.4 (q), 195.6 (s), and 96.1 (d). In the HMBC spectrum, the correlations of δH 2.11 (3H, s) with δC 195.6 (s) and 96.1 (d) established a CH3− CO−CH− unit among C-24, C-23, and C-22, while the HMBC correlation of δH 5.09 (1H, s, H-22) with δC 150.7 (s, C-3) suggested the unit to be connected to C-3. ROESY correlations of H-9/H-21, H-21/H-19, and H-19/H-16 indicated that the relative configuration of 5 was also the same as that of venalstonine. Detailed analysis of 2D NMR data (HSQC, HMBC, 1H−1H COSY, ROESY) established the structure of 5 to be as shown, and it was named melodinine Q. Melodinine R (6) was obtained as a colorless oil. The molecular formula C24H28N2O4 was established by HREIMS, 57 Da higher than that of 19-(R)-hydroxytabersonine.23 Compound 6 was readily identified as an acetonyl derivative of 19-(R)-hydroxytabersonine by the carbon resonances at δC 46.2 (t, C-22), 207.3 (s, C-23), and 30.4 (q, C-24), which was placed at C-3 on
Table 1. 1H NMR Data of 1−4α (δ in ppm and J in Hz) no.
1
2
3
4
9.20, s 3.09, overlap 3.41, dd (15.6, 3.6)
5a 5b
9.32, s 3.00, d (15.6) 3.38, ddd (15.6, 4.8, 1.2) 2.99, overlap 2.32, m
9.16, s 3.17, d (10.2) 3.42, ddd (10.2, 4.8, 1.5) 2.70, m 3.00, t (7.8)
6a
1.67, m
6b
1.74, dd (12.6, 4.8) 6.90, d (9.6) 6.20, dd (9.6, 1.2)
1.63, dd (10.8, 3.6) 1.93, m
9.27, s 3.17, d (15.6) 3.41, ddd (15.6, 4.8, 1.2) 2.76, m 2.98, m, overlap 1.69, dd (11.4, 4.2) 1.95, m
7.12, s
7.14, s
1.73, dd (11.4, 3.6) 2.97, m, overlap 6.74, d (2.4)
6.60, s 5.75, dd (10.2, 3.6)
6.59, s 5.77, ddd (9.9, 4.8, 1.2)
6.62, dd (7.8, 2.4) 6.86, d (7.8) 5.78, ddd (9.6, 4.8, 1.5)
5.66, d (10.2) 2.43, d (15.0) 2.48, d (15.0) 0.59, t (7.2) 0.79, m 0.96, m 2.56, s 2.08, s 3.66, s
5.68, d (9.9)
5.70, d (9.6)
2.44, d (14.4)
2.44, d (15.0)
2.50, d (14.4)
2.50, d (15.0)
0.68, 0.83, 0.98, 2.62, 4.43, 3.68, 3.33,
0.63, 0.84, 0.98, 2.62,
N1-H 3a 3b
9 10
2.64, m 2.94, overlap
11 12 14 15
5.56, d (1.2) 5.80, ddd (10.2, 4.8, 1.2) 5.62, d (10.2)
17a
2.43, s
17b 18 19a 19b 21 22 OOCMe OMe α
0.79, t (7.2) 1.44, q (7.2) 2.66, s 3.78, s
t (7.5) m m s s s s
t (7.8) m m s
3.67, s
Compound 1 was measured in CDCl3; 2, 3, and 4 in acetone-d6.
compound 1 displayed 21 carbon resonances assigned to two methyl, five methylene, six methine, and eight quaternary carbons (Table 3). These data suggested that 1 was an Aspidospermatype alkaloid related to 11-hydroxytabersonine (10) with identical rings B−E (Figure S1, Supporting Information).11,21 A significant difference was that the benzene ring A was oxidized to a dienone system, as deduced from three deshielded olefinic protons at δH 6.90 (d, J = 9.6 Hz), 6.20 (dd, J = 9.6, 1.2 Hz), and 5.56 (d, J = 1.2 Hz),22 as well as the carbonyl carbon resonance at δC 186.2. The dienone system was further determined as 9(10),12(13)-dien-11-one by HMBC and 1H−1H COSY correlations (Figure S1, Supporting Information). In addition, C-8 was an oxygenated quaternary carbon at δC 74.9, on the basis of the HMBC correlations from H-6, H-10, H-12, and N-H to C-8. The relative configuration of 1 was assigned on the basis of the ROESY experiment measured in DMSO (Figure S1, Supporting Information). The ROESY correlations of OH-8/H-21 and H-21/H-19 indicated the α-orientation of 221
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Table 2. 1H NMR Data of 5−9α (δ in ppm and J in Hz) no. 1 3a 3b 5a 5b 6a 6b 9 10 11 12 14 15 16 17a 17b 18a 18b 19a 19b 21 22a 22b 24 OOCMe α
5
3.37, 3.59, 1.40, 2.66, 7.03, 6.76, 7.06, 6.72, 7.65, 5.98, 2.92, 1.57, 2.27, 1.30, 1.91, 1.48, 1.79, 3.56, 5.09,
m m dd (12.8, 5.6) m d (7.6) t (7.6) t (7.6) d (7.6) d (10.0) d (10.0) t (9.8) d (10.2) m t (11.6) m t (12.0) m s s
2.11, s 3.72, s
6
7
9.38, s 4.01, dd (13.6, 6.0)
9.34,s 4.06, dd (13.0, 5.5)
7.95 3.23, m
3.00, 3.11, 1.91, 2.02, 7.35, 6.86, 7.13, 7.03, 5.94, 5.81,
2.78, 2.83, 1.63, 1.83, 7.22, 6.84, 7.13, 7.03, 3.56, 3.16,
3.10, 3.18, 1.98, 2.52, 7.42, 7.09, 7.19, 6.71, 5.92, 5.99,
m dd (15.0, 6.9) m overlap d (7.5) t (7.5) t (7.5) d (7.5) dd (10.2, 4.8) d (10.2)
m m dd (11.0, 4.0) m d (7.5) t (7.5) t (7.5) d (7.5) t (4.2) d (4.2)
2.12, d (15.0) 3.06, d (15.0) 0.89, d (6.0)
2.36, d (14.5) 2.57, d (14.5) 0.72, t (7.0)
3.27, q (6.0)
0.84, 1.04, 2.74, 2.96,
3.21, 2.68, 2.88, 2.22, 3.66,
s dd (13.6, 6.0) overlap s s
8
9
dd (16.8, 9.0) m m overlap d (7.8) t (7.8) t (7.8) d (7.8) dt (10.2, 3.6) d (10.2)
2.51, d (13.8) 3.36, d (13.8) 2.23, s
m m s m
3.89, 3.99, 4.02, 3.49, 2.07, 3.31, 7.53,
m m m t (10.2) dd (14.7, 8.7) overlap d (2.4)
6.66, 6.72, 5.69, 5.91,
dd (8.4, 2.4) d (8.4) m dd (10.5, 2.7)
2.50, d (13.8) 2.97, d (13.8) 5.07, dd (17.4, 10.8) 5.83, dd (17.4, 10.8)
2.23, s 3.70, s
4.18, s
3.89, s
3.57, s
3.57, s
Compounds 5 and 8 were measured in CDCl3; 6 and 7 in acetone-d6; 9 in methanol-d4.
Table 3. 13C NMR Data of 1−9α (δ in ppm and J in Hz)
α
no.
1
2
3
4
5
6
7
8
9
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 OOCMe OOCMe OMe
158.5 C 51.2 CH2 51.7 CH2 36.9 CH2 56.9 C 74.9 C 138.9 CH 132.8 CH 186.2 C 100.3 CH 164.5 C 125.0 CH 134.1 CH 98.6 C 29.5 CH2 8.2 CH3 27.1 CH2 44.1 C 63.3 CH
168.0 C 51.1 CH2 51.2 CH2 45.7 CH2 55.6 C 130.4 C 123.8 CH 120.4 C 155.0 C 98.8 CH 143.6 C 125.9 CH 133.6 CH 92.0 C 29.3 CH2 7.7 CH3 27.4 CH2 42.3 C 70.6 CH 30.5 CH3
167.8 C 51.1 CH2 51.3 CH2 45.8 CH2 55.6 C 129.9 C 123.0 CH 116.9 C 156.5 C 98.7 CH 145.1 C 126.1 CH 133.5 CH 92.3 C 29.3 CH2 7.7 CH3 27.4 CH2 42.2 C 70.8 CH 71.0 CH2
168.0 C 51.0 CH2 51.4 CH2 45.6 CH2 56.5 C 140.3 C 110.5 CH 152.8 C 114.3 CH 110.9 CH 137.1 C 126.0 CH 133.5 CH 91.0 C 29.4 CH2 7.7 CH3 27.5 CH2 42.2 C 70.8 CH
168.7 C 51.0 CH3 58.0 CH3
168.8 C 50.9 CH3
167.7 C 51.0 CH 48.0 CH2 45.0 CH2 56.1 C 138.6 C 122.4 CH 121.3 CH 128.6 CH 110.6 CH 144.5 C 57.2 CH 59.0 CH 90.5 C 24.1 CH2 7.4 CH3 25.7 CH 41.6 C 63.3 CH 38.0 CH2 207.7 C 30.5 CH3 168.5 C 51.1 CH3
167.7 C 62.7 CH2 66.5 CH2 34.7 CH2 60.4 C 127.9 C 117.7 CH 154.8 C 116.2 CH 117.5 CH 129.5 C 117.5 CH 132.5 CH 63.5 C 43.3 CH2 115.5 CH2 142.3 CH 50.5 C 92.9 CH
168.7 C 50.8 CH3
165.7 C 53.2 CH 52.0 CH2 44.1 CH2 56.9 C 139.5 C 122.8 CH 121.5 CH 128.5 CH 110.3 CH 144.7 C 129.3 CH 130.3 CH 91.2 C 30.2 CH2 19.1 CH3 67.5 CH 43.9 C 61.8 CH 46.2 CH2 207.3 C 30.4 CH3 168.9 C 50.9 CH3
166.6 C 46.2 CH2 52.1 CH2 35.6 CH2 58.3 C 128.7 C 126.5 CH 124.1 CH 127.8 CH 115.5 CH 134.7 C 127.9 CH 126.7 CH 62.3 C 41.5 CH2 25.4 CH3 207.5 C 55.5 C 74.9 CH
168.9 C 51.8 CH3
66.6 C 150.7 C 46.2 CH2 34.8 CH2 56.0 C 136.8 C 121.0 CH 119.7 CH 127.8 CH 111.5 CH 148.9 C 122.8 CH 139.7 CH 43.1 CH 27.4 CH2 32.4 CH2 28.5 CH2 33.7 C 64.0 CH 96.1 CH 195.6 C 31.4 CH3 173.3 C 52.2 CH3
169.7 C 53.1 CH3
170.6 C 53.4 CH3
Compounds 1, 5, and 8 were measured in CDCl3; 2, 3, 4, 6, and 7 in acetone-d6; 9 in methanol-d4.
the basis of HMBC correlations of δH 2.68 (1H, dd, J = 13.6, 6.0 Hz, H-22a) and 2.88 (1H, overlap, H-22b) with δC 53.2 (d, C-3), as well as the 1H−1H COSY cross-peak between H-22
and H-3. ROESY correlations of H-22/H-21 indicated the β-orientation of H-3. Thus, the structure of melodinine R (6) was determined as shown. 222
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water-soluble material, adjusted to pH 9−10 with 10% ammonia solution, was extracted with EtOAc to give an alkaloidal extract (42 g). The extract was subjected to a silica gel column (CHCl3−Me2CO, 1:0 to 0:1) to afford fractions I−VIII. Fraction I (4.4 g) was separated by silica gel CC (petroleum ether−EtOAc, 20:1−5:1) to afford 11 (223 mg) and 12 (1870 mg). Fraction II (3.3 g) was subjected to MPLC with RP-18 CC (MeOH−H2O, 6:4−10:0), followed by silica gel CC (petroleum ether−Me2CO, 15:1−5:1), to yield 15 (32 mg), 16 (37 mg), and a mixture. The mixture was chromatographed on a silica gel column (petroleum ether−EtOAc, 10:1−6:1) to afford 5 (13 mg) and 13 (63 mg). Fraction III (12.5 g) was separated by silica gel CC (petroleum ether−Me2CO, 8:1 to 2:1), then by RP-18 CC, eluted with MeOH−H2O (5:5−10:0), to afford venalstonine (220 mg), scandine (6560 mg), and a mixture. The latter was purified by Sephadex LH-20 (CHCl3−MeOH, 1:1) to give 7 (6 mg) and 3-oxo-tabersonine (23 mg). Fraction IV (2.8 g) was subjected to MPLC with RP-18 CC (MeOH−H2O, 4:6−8:2) to give subfractions IV-a and IV-b. Subfraction IV-a was further separated by silica gel CC (petroleum ether− Me2CO, 4:1) to yield 3 (8 mg) and 14 (28 mg). Subfraction IV-b was subjected to Sephadex LH-20 CC (CHCl3−MeOH, 1:1), then silica gel CC (petroleum ether−Me2CO, 6:1), to give 4 (7 mg) and 6 (4 mg). Fraction V (3.9 g) was separated by silica gel CC (CHCl3− MeOH, 15:1) to yield 10 (1630 mg) and a mixture. Further separation of the mixture by RP-18 CC (MeOH−H2O, 5:5) yielded 2 (5 mg). Separation of fraction VI (1.7 g) by RP-18 CC, eluted with MeOH− H2O (3:7−8:2), and then by silica gel CC (CHCl3−MeOH, 10:1) afforded 1 (62 mg) and 8 (12 mg). Fraction VII (2.5 mg) was separated by RP-18 CC (CH3OH−H2O, 2:8−5:5), then further by Sephadex LH20 CC (MeOH), to yield 9 (55 mg) and 10-hydroxyscandine (432 mg). Melodinine M (1): yellow needles (MeOH); mp 109−111 °C; [α]26D −66.3 (c 0.102, MeOH); UV (MeOH) λmax (log ε) 370 (4.00), 282 (4.01), 223 (4.27), 203 (4.23) nm; IR (KBr) νmax 3431, 2932, 1729, 1656, 1633, 1610, 1582, 1437, 1382, 1308, 1253, 1158, 1049, 745 cm−1; 1H (400 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 3; positive ion HRESIMS m/z 369.1811 (calcd for C21H25N2O4 [M + H]+, 369.1814). Melodinine N (2): white powder; mp 87 °C; [α]25D −146.3 (c 0.231, MeOH); UV (MeOH) λmax (log ε) 333 (3.94), 255 (4.02), 203 (4.22) nm; IR (KBr) νmax 3439, 2958, 2925, 1669, 1487, 1438, 1264, 1155, 1102, 1058, 576 cm−1; 1H (400 MHz) and 13C NMR (125 MHz) data (Me2CO-d6), see Tables 1 and 3; HREIMS m/z 366.1927 (calcd for C22H26N2O3 [M]+, 366.1943). Melodinine O (3): white powder; mp 95−96 °C; [α]25D −186.3 (c 0.095, MeOH); UV (MeOH) λmax (log ε) 330 (4.11), 263 (4.44), 202 (4.36) nm; IR (KBr) νmax 3430, 2930, 1722, 1675, 1620, 1439, 1264, 1212, 1105, 1058, 576 cm−1; 1H (500 MHz) and 13C NMR (150 MHz) data (Me2CO-d6), see Tables 1 and 3; HREIMS m/z 396.2041 (calcd for C23H28N2O4 [M]+, 396.2049). Melodinine P (4): colorless oil; [α]24D −158.3 (c 0.164, MeOH); UV (MeOH) λmax (log ε) 342 (3.87), 313 (3.98), 204 (4.23) nm; IR (KBr) νmax 3427, 2960, 2926, 1669, 1613, 1468, 1439, 1382, 1273, 1189, 1111, 809, 582 cm−1; 1H (400 MHz) and 13C NMR (150 MHz) data (Me2CO-d6), see Tables 1 and 3; HREIMS m/z 352.1782 (calcd for C21H24N2O3 [M]+, 352.1787). Melodinine Q (5): colorless needles (MeOH); mp 136−138 °C; [α]24D +186.8 (c 0.048, MeOH); UV (MeOH) λmax (log ε) 362 (4.13), 239 (4.00), 206 (4.25) nm; IR (KBr) νmax 3440, 2951, 2925, 1718, 1640, 1614, 1520, 1449, 1324, 1216, 1167, 961, 753 cm−1; 1H (400 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 3; positive ion HRESIMS m/z 391.2027 (calcd for C24H27N2O3 [M + H]+, 391.2021). Melodinine R (6): colorless oil; [α]24D −42.9 (c 0.104, MeOH); UV (MeOH) λmax (log ε) 328 (3.84), 277 (4.05), 203 (4.34) nm; IR (KBr) νmax 3431, 2924, 1711, 1678, 1632, 1610, 1466, 1438, 1246, 1103, 900, 748 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data (Me2CO-d6), see Tables 2 and 3; HREIMS m/z 408.2050 (calcd for C24H28N2O4 [M]+, 408.2049). Melodinine S (7): colorless needles (Me2CO); mp 73−75 °C; [α]26D −331.7 (c 0.100, MeOH); UV (MeOH) λmax (log ε) 328 (4.33), 299 (4.18), 225 (4.17), 202 (4.23) nm; IR (KBr) νmax 3440,
Melodinine S (7) gave the molecular formula C24H28N2O4, indicating 12 degrees of unsaturation. The 1D NMR data showed that 7 had a structure similar to that of 3α-acetonyltabersonine (16)15 except for the 14/15 epoxy group (δC 57.2, 59.0) in 7. The suggestion was supported by the molecular formula and HMBC correlations of δH 3.56 (1H, t, J = 4.2 Hz, H-14) with C-3 and C-22 and of δH 3.16 (1H, d, J = 4.2 Hz, H-15) with C-21, C-20, C-17, and C-19. ROESY correlations of H-21/H-19, H-22/H-21, H-3/H-14, and H-14/H-15 placed the 3α-acetonyl and the 14, 15-epoxy ring on the same side of the molecule. Detailed analysis of 2D NMR data (HSQC, HMBC, ROESY) established the structure of melodinine S (7) to be as shown. Melodinine T (8) possessed the molecular formula C21H22N2O4. The 1H and 13C NMR data (Tables 2 and 3) were very similar to those of scandine18 except that the terminal double bond between C-19 and C-18 was oxidized to be an acetyl at δC 207.5 (s) and 25.4 (q), as supported by the HMBC correlations of δH 2.23 (3H, s, H-18) with δC 207.5 (s, C-19) and 55.5 (s, C-20). ROESY correlations indicated that the relative configuration of 8 was the same as that of scandine. Melodinine U (9) was isolated as a white, amorphous powder (C21H22N2O5 by HREIMS), 16 Da higher than that of 10hydroxyscandine.19 Compound 9 was readily identified as 10hydroxyscandine-N(4)-oxide from 1H and 13C NMR data, in particular the characteristic downfield shifts of the carbon resonances at δC 62.7, 66.5, and 92.9 for C-3, C-5, and C-21, respectively, with respect to those of 10-hydroxyscandine.7c−7d All alkaloids were evaluated for their cytotoxicity against five human cancer cell lines using the MTT method reported previously.24 Compounds 6, 11, and 16 exhibited stronger inhibitory effects against five human cancer cell lines with lower IC50 values than those of cisplatin. Compounds 2, 3, 7, 12, 13, and 14 displayed moderate cytotoxicity against one or more of the cell lines. The other compounds were considered to be noncytotoxic, with IC50 values greater than 10 μM. It is noteworthy that tabersonine derivatives with an acetonyl moiety at C-3 inhibit five human cancer cell lines significantly in comparison with tabersonine.
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrometer. IR spectra were obtained by a Bruker FT-IR Tensor 27 spectrometer using KBr pellets. 1D and 2D NMR spectra were run on an AVANCE III-600 MHz or a Bruker DRX-500 MHz spectrometer or an AV-400 MHz spectrometer with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to solvent signals. HREIMS was recorded on a Waters Auto Premier P776 spectrometer. HRESIMS was recorded on an API QSTAR Pulsar I spectrometer. Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People’s Republic of China), RP-18 gel (20−45 μm, Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Sweden). Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd. Qingdao), and spots were visualized by Dragendorff’s reagent. Plant Material. M. suaveolens was collected from Luchun County, Yunnan Province, P. R. China, and identified by Dr. Chun-Xia Zeng, Kunming Institute of Botany. A voucher specimen (No. Zeng20091026) has been deposited at Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. An air-dried and powdered sample (16 kg) was extracted with 90% MeOH (24 h × 4). The extract was partitioned between EtOAc and a 0.5% HCl solution. The acidic 223
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Journal of Natural Products
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3375, 2961, 2901, 1704, 1669, 1608, 1465, 1439, 1294, 1243, 1113, 757 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (Me2COd6), see Tables 2 and 3; positive ion HRESIMS m/z 409.2122 (calcd for C24H29N2O4 [M + H]+, 409.2127). Melodinine T (8): white powder; mp 75−76 °C; [α]26D +204.0 (c 0.100, MeOH); UV (MeOH) λmax (log ε) 396 (1.44), 261 (3.96), 208 (4.54) nm; IR (KBr) νmax 3432, 2924, 1676, 1617, 1487, 1455, 1266, 1156, 1106, 738 cm−1; 1H (400 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 3; positive ion HRESIMS m/z 367.1449 (calcd for C21H23N2O4 [M + H]+, 367.1446). Melodinine U (9): white powder; mp 130−132 °C; [α]26D +177.3 (c 0.103, MeOH); UV (MeOH) λmax (log ε) 306 (3.59), 271 (3.96), 205 (4.39) nm; IR (KBr) νmax 3425, 3235, 2955, 1735, 1672, 1617, 1503, 1469, 1251, 1181, 1138, 973, 732 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data (MeOH), see Tables 2 and 3; positive ion HRESIMS m/z 383.1613 (calcd for C21H23N2O5 [M + H]+, 383.1606).
ACKNOWLEDGMENTS The authors are grateful to the Natural Science Foundation of China (31170334, 21072198), the National Basic Research Program of China (973 Program 2009CB522300), and Chinese Academy of Sciences (KSCX2-EW-R-15) for partial financial support.
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compd
HL-60
SMMC-7721
A-549
MCF-7
SW480
3.5 10.0 28.1 0.7 4.8 6.0 0.5 4.5 5.8 6.3 15.5 0.2 1.3
15.5 15.2 >40 3.3 11.2 15.5 1.1 5.6 6.0 16.0 24.7 0.3 14.6
18.5 18.2 >40 3.9 15.2 17.6 1.0 14.7 15.5 19.7 >40 0.6 10.7
15.0 15.7 >40 1.8 7.0 >40 0.2 9.9 14.2 21.3 >40 0.4 18.4̀
15.1 14.9 >40 1.6 13.5 >40 2.4 12.1 15.0 13.1 >40 0.5 17.7
Cytotoxicity Assays. Five human cancer cell lines, human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7, and colon cancer SW480 cells, were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The assays were performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) method in 96-well microplates.24 Briefly, 100 μL of adherent cells was seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the test compound at concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control. After each compound treatment, cell viability was detected and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method.25
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ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR and MS spectra of melodinines M−U (1−9). These materials are available free of charge via the Internet at http://pubs.acs.org.
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Table 4. Cytotoxicity of Compounds 2−4, 6, 7, and 10−16 (IC50, μM) 2 3 4 6 7 10 11 12 13 14 15 16 cisplatin
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